大鼠致畸试验中阳性对照环磷酰胺的给药方法研究

目的： 比较致畸敏感期环磷酰胺不同给药方案对孕鼠的致畸效应,确定一种最佳的给药方案以指导环磷酰胺作为大鼠致畸敏感期生殖毒性试验阳性对照的使用。 方法：交配成功的SD大鼠随机分为3组,每组10只,分别为：环磷酰胺连续给药组 (A组),于妊娠第6~15天 (即GD6～GD15)每天按4.8 mg/kg皮下注射给予环磷酰胺;环磷酰胺单次给药组 (B组),于GD12一次性按12 mg/kg皮下注射给予环磷酰胺;对照组 (C组),于GD6～GD15每天皮下注射给予大鼠相应体积的生理盐水。孕期称取母鼠体质量并观察一般状况,于GD20用CO2对孕鼠实施安乐死,解剖后称重并记录其生殖器官质量,检查胎仔外观、骨骼和内脏畸形情况。 结果：两个环磷酰胺给药组一般状况观察均未见异常;B组子宫胎盘质量、胎盘质量、子宫质量低于对照组 (P<0.05)。 外观检查,A组未出现显著的畸形种类;B组能引起明显的外观畸形,主要有脑膜膨出、小头、脑露、足内翻等。内脏检查,A组显著增加的畸形仅见侧脑室扩大或淤血;B组出现明显的内脏畸形,主要表现为第三脑室扩大或淤血、侧脑室扩大或淤血、心室心房异常等。骨骼检查,A组骨骼畸形种类较少,且与对照组比较差异无统计学意义;B组大部分骨骼畸形发生率比对照组高,主要表现在头部和胸腔,如基蝶骨异常、肋骨数目异常等。结论：在本试验条件下,大鼠于GD12按12 mg/kg经皮下注射给予环磷酰胺为较为理想的阳性对照给药方法,显示出更宽的畸形谱和更重的畸形程度,推荐在大鼠致畸敏感期生殖毒性试验中选用。     

含铜宫内节育器的致畸作用评价

目的：评价含铜宫内节育器对SD大鼠的致畸作用。方法：试验设4组,分别为溶剂对照组（生理盐水）、给药组含铜宫内节育器浸提液,浸提比例分别为0.1、0.3和0.9g/ml）,每组24只孕鼠。各组于妊娠期第6～15天静脉注射给予受试物,于孕第20天时处死孕鼠,观察孕鼠一般状况、体质量、活胎数、胎鼠外观、骨骼及内脏等。结果：受试组孕鼠孕期的体质量增长、窝质量、黄体数、着床数、活胎数、胎鼠的身长、尾长等指标与溶剂对照组相比,差异均无统计学意义（P〉0.05）。但0.9g/ml组的吸收胎率（7.3%）较对照组（3.4%）明显升高（P〈0.05）。胎鼠外观、内脏及骨骼检查均未见畸形。结论：在本实验条件下,SD大鼠静脉注射含铜宫内节育器样品浸提液（0.1～0.9g/ml）,仅0.9g/ml时有早期胚胎发育毒性,各浓度均未见母体毒性与致畸性。     

Human placental aryl hydrocarbon hydroxylase: studies with fluorescence histochemistry.

Tissue sections from human placentas taken at term were studied after time-sequential incubations with benzo[a]pyrene and appropriate cofactors for mixed-function oxidation. Fluorescence microscopy revealed that the enzymic reaction appeared to be most active in the syncytial trophoblast, though the fluorescence of hydroxylated metabolites also could be observed in other placental cell types. A comparison of sections from placentas with very low versus very high aryl hydrocarbon hydroxylase activities provided evidence that induction of the human placental enzyme system with pol7cyclic aromatic hydrocarbons also appeared to occur primarily in the syncytium. When considered in conjunction with previous studies on human placental aryl hydrocarbon hydroxylase, the results tended to indicate that fetal elements of the human placenta contain the necessary electron-transport components for catalysis of mixed-function oxidations of chemical carcinogens and other foreign compounds and that this hydroxtlase system is readily inducible in the same fetal cells by components present in cigarette smoke.     

先天性腭裂形成中胚胎腭器官的电镜观察

背景与目的： 建立先天性腭裂模型观察其形成过程中胚胎腭突细胞的超微结构改变。 材料与方法： 于NIH雌性小鼠受孕第12.5 d(GD12.5)通过腹腔注射磷酸地塞米松(50 mg/kg)诱发NIH小鼠胚胎先天性腭裂模型,同时设正常对照组注射生理盐水相同条件下饲养。实验组和对照组母鼠分别于GD13.5、GD14.5、GD15.5脱颈处死,剖腹取出胎鼠,切取胎鼠头部制作光镜和电镜样本,采用扫描电镜和透射电镜观察胚胎腭发育及先天性腭裂形成过程中腭突细胞的超微结构。 结果： 随着胚胎发育,对照组腭突双侧裂隙逐渐变小,上皮基底膜破坏,腭突中嵴上皮细胞(MEE)细胞核染色质呈块状并边集,并可见上皮细胞内出现分泌物质,胚腭间充质细胞(EPM)细胞之间基质丰富,胚胎GD15.5腭突完全融合;实验组腭突生长缓慢,体积较同期对照组小,随着腭突的发育,腭突表层MEE细胞仍然连接紧密,基底膜完整,上皮细胞多层化,细胞表面出现纤毛,EPM细胞之间基质较少,在GD15.5形成裂隙。 结论： 地塞米松作用后胚胎腭突细胞的正常发育分化受到影响,从而导致腭裂形成。     

Type-C virus particles in placenta of the cottontop marmoset (Saguinus oedipus).

Electron microscopy of near-term placentas of two cottontop marmosets (Saguinus oedipus) revealed, in one placenta, the presence of budding and mature C-type virus particles associated with the basal trophoblast. The particles were morphologically similar to those observed by other investigators in placentas of other primate species.     

Hsp60基因在小鼠腭和肢正常与异常发生过程中的表达

背景与目的:研究热休克蛋白60基因(heat shock protein 60 gene,Hsp60)在小鼠胚胎腭、前肢正常和异常发育过程中的表达情况.材料与方法:将受孕后ICR小鼠随机分为实验组和对照组2组,每组各64只,于孕10 d(gestational day 10,GD10),分别经口一次给予实验组孕鼠80 mg/kg的全反式视黄酸,对照组孕鼠给予等体积的大豆油,并分别于GD11～GD18取2组胎鼠的前肢,于GD15～GD17取2组胎鼠的腭,利用实时荧光定量PCR检测Hsp60的表达丰度.结果:Hsp60在正常、异常肢和腭中均有表达.对照肢的表达丰度在GD14和出生前呈高表达,而实验肢的表达在各胚龄无明显差异(P＞0.05);实验肢Hsp60在GD11～GD18的表达水平高于同一胚龄的对照肢(P＜0.05).在正常腭中,Hsp60恒定表达,在异常腭中,Hsp60的表达随胚龄增大而降低;在GD15～GD17的表达丰度为实验腭低于同胚龄的对照腭(P＜0.05).结论:全反式视黄酸所致的短肢中,脚60的表达呈应激性升高;而在异常腭中,Hsp60的表达受到抑制.     

Influence of clamping-induced ischemia and reperfusion on the response of a mouse melanoma to radiation and hyperthermia.

PURPOSE: To study the response of a mouse melanoma to radiation and hyperthermia under acute hypoxia and reperfusion. MATERIALS AND METHODS: B16F1 melanoma of 100+/-10 mm3, in C57BL mouse, were locally exposed to 10Gy gamma radiation (RT), 43 degrees C for 30 min (HT) in a water bath, or RT followed immediately by HT, under clamping (acute hypoxia) or 1 h after reperfusion. Tumour regression, volume doubling time (VDT), growth delay (GD), apoptosis and microvascular density (MVD) were studied. RESULTS: Under clamping, HT increased the VDT and GD to > 20 days above control and resulted in > 50% regression (PR) in all the tumours, whilst RT + HT synergistically enhanced VDT and GD. Under reperfusion, HT produced 25% PR against 16% by RT, with no increase in VDT and GD compared to RT. RT + HT significantly enhanced VDT and GD above that of RT or HT, but did not further increase PR of reperfused tumours. HT under clamping caused > 50% increase in apoptic cells over control and decreased MVD to 1/3rd of control. RT + HT further enhanced apoptotic cells to > 70% and reduced MVD to 1/6th of control. CONCLUSIONS: These results suggest that combination of radiotherapy with hyperthermia could benefit treatment of tumours with ischemia-induced acute hypoxia.     

Morphologic aspect of the placenta in young and adult pregnant rats bearing Walker 256 carcinoma

In the present study we investigated the influence of Walker 256 tumor growth on the modification of placental morphology and on fetal development in young and adult pregnant rats. After mating, female rats were divided into six groups: young control pregnant (Y), young pregnant with tumor (Yw), young pregnant injected with ascitic fluid (Ya), adult control pregnant (A), adult pregnant with tumor (Aw), and adult pregnant injected with ascitic fluid (Aa). Rats from tumor-bearing groups (Yw and Aw) were injected with 2.5 x 10(6) viable tumor cells into the right flank. Rats from Ya and Aa groups received daily inoculations of ascitic fluid (2.0 ml, i.p.) obtained from tumor-bearing rats without tumor cells. After 21 days, all animals were killed and the placentas were weighed and fixed with paraformaldehyde for histological analysis. Compared with control groups (Y and A), both tumor-bearing groups (Yw and Aw) presented the following changes: i) hemorrhage in the decidua and in the trophoblast giant cell layer; ii) disarrangement of the spongy zone, iii) restricted delimitation of the maternal and fetal blood vessels in the placental labyrinth; iv) hemorrhage and edema in the placental labyrinth. Similar results were observed in the placenta of groups injected with ascitic fluid (Ya and Aa). These results indicate that tumor development during pregnancy can have deleterious effects on placenta and fetus. These observations extend our previous data of extensive fetal reabsorption in both pregnant tumor-bearing and ascitic fluid-injected animals. These changes in placental morphology may be related to the synthesis and release of some factors by the tumor and the host cells, which could act directly or indirectly on placental tissue.     

Influence of clamping-induced ischemia and reperfusion on the response of a mouse melanoma to radiation and hyperthermia

Purpose: To study the response of a mouse melanoma to radiation and hyperthermia under acute hypoxia and reperfusion. Materials and methods: B16F1 melanoma of 100 ± 10mm³, in C57BL mouse, were locally exposed to 10 Gy gamma radiation (RT), 43°C for 30 min (HT) in a water bath, or RT followed immediately by HT, under clamping (acute hypoxia) or 1 h after reperfusion. Tumour regression, volume doubling time (VDT), growth delay (GD), apoptosis and microvascular density (MVD) were studied. Results: Under clamping, HT increased the VDT and GD to > 20 days above control and resulted in > 50% regression (PR) in all the tumours, whilst RT + HT synergistically enhanced VDT and GD. Under reperfusion, HT produced 25% PR against 16% by RT, with no increase in VDT and GD compared to RT. RT + HT significantly enhanced VDT and GD above that of RT or HT, but did not further increase PR of reperfused tumours. HT under clamping caused > 50% increase in apoptic cells over control and decreased MVD to 1/3rd of control. RT + HT further enhanced apoptotic cells to > 70% and reduced MVD to 1/6th of control. Conclusions: These results suggest that combination of radiotherapy with hyperthermia could benefit treatment of tumours with ischemia-induced acute hypoxia.     

Immunologic considerations of fetal survival in mice

Studies were done on the effects on pregnancy in DBA/2 mice of sensitization prior to pregnancy with certain tissue antigens. Tissues used were paternal testes, spleen and kidney, weanling offspring spleen and kidney, placenta, and embryo. With the exception of the mice receiving placental inoculations, the DBA/2 females did not demonstrate any altered rate of pregnancy, size of litter, or viability of offspring. Female mice receiving placental inoculations showed a slightly decreased rate of pregnancy, although this was not statistically significant. Histological examination of placentas and implantation sites from sensitized mice sacrificed during the second trimester of pregnancy revealed no evidence of immune rejection phenomena. Examination of lung tissue from term pregnant DBA/2 females failed to reveal the presence of trophoblastic emboli and suggests that a strict anatomical separation of mother and fetus occurs in the mouse. Full-thickness allografts to pregnant DBA/2 female mice showed a very slightly accelerated rate of rejection in those animals bearing fetuses with a genetic background similar to the donor of the graft. This slightly accelerated rate of skin graft rejection suggests that paternal antigens present in the conceptus can elicit a detectable immune response in the mother under certain conditions.     

重组人B淋巴细胞刺激因子受体-抗体融合蛋白(RCT-18)对大鼠的致畸作用

目的:观察重组人B淋巴细胞刺激因子受体-抗体融合蛋白(RCT-18)对大鼠胚胎及胎仔的发育毒性和致畸作用。方法:采用雌性SD大鼠,交配成功后随机分为4组,即RCT-18高(129 mg/kg)、中(37 mg/kg)、低(11 mg/kg)剂量组及阴性对照(0.9%NaCl注射液)组,每组≥25只,于妊娠第6～15天连续5次(隔天1次)经皮下注射给药。实验过程中观察动物的一般反应、体质量、摄食量变化,于妊娠第20天解剖孕鼠,对着床、吸收胎、死胎、活胎、黄体进行计数,对胎仔的体质量、身长、尾长,以及外观形态、内脏和骨骼发育等指标进行评价。结果:孕鼠、胚胎形成、胎仔生长发育、外观形态、内脏和骨骼发育等各项指标均无明显异常,与阴性对照组比较无明显毒性影响(P0.05)。结论:在本试验条件下,RCT-18对SD大鼠未见明显母体毒性和胚胎-胎仔发育毒性,无致畸作用。     

电子垃圾污染区新生儿胎盘镉含量及金属硫蛋白表达水平

目的：检测贵屿电子垃圾污染区新生儿胎盘镉含量及胎盘金属硫蛋白（metallothionein,MT）表达量,评估贵屿地区新生儿镉暴露情况及对新生儿的可能影响。方法：选取贵屿当地医院妇产科2006年7~9月出生的足月健康新生儿胎盘100例为实验组,纳入研究的产妇为贵屿镇当地居民,妊娠期间在贵屿居住。取汕头市潮南民生医院妇产科2006年5~6月出生的足月健康新生儿胎盘52例为对照组,产妇来自贵屿周边乡镇。石墨炉原子吸收法检测胎盘镉含量。链霉菌素_生物素（S_P）免疫组化技术检测胎盘组织MT的表达水平。问卷调查收集可能影响镉负荷的产妇年龄、家庭、环境、健康、饮食等因素。结果：实验组新生儿胎盘镉水平的平均值为（0.17±0.48）μg/g,明显高于对照组（0.10±0.11）μg/g,差异具有统计学意义（P〈0.01）。相关分析表明产妇在贵屿居住时间、产妇妊娠期间在贵屿居住时间、产妇怀孕期间在公路附近活动时间是影响胎盘镉水平的主要因素。S_P免疫组织化学检测显示胎盘组织中蜕膜细胞、合体滋养层细胞、绒毛间质细胞均有MT的表达。实验组胎盘组织MT阳性表达率（67.00%）高于对照组（32.69%）（P〈0.01）。新生儿胎盘MT表达量与胎盘镉水平呈显著正相关（r=0.761,P〈0.05）。结论：贵屿部分新生儿处于高镉负荷状态,贵屿当地环境和从事电子垃圾作业是影响当地新生儿高镉负荷的危险因素。贵屿地区新生儿胎盘可能通过增加MT的表达拮抗镉的毒性。     

Role of metallothionein in human placenta and rats exposed to cadmium.

A strong positive relationship between zinc and copper and metallothionein (MT) in placentas has been found, but a negative one between cadmium and MT. In rats given cadmium i.p. as the chloride, liver cadmium is lower and kidney cadmium higher than in cadmium-treated non-pregnant rats, suggesting that pregnancy enhances mobilization of cadmium from liver to kidney. The cadmium concentration of digested whole fetuses is not significantly increased in offspring of dams given cadmium i.p. as the chloride or CdMT, but the placental levels of cadmium and MT are increased. The placenta therefore acts as a barrier to maternal-fetal cadmium transfer. The way in which cadmium is retained in the placenta but zinc and copper are transferred to the fetus is not understood, since all are bound to MT. Focal renal tubular necrosis and placental necrosis occur at the same level of cadmium exposure, suggesting a similar threshold to cadmium toxicity for the two organs.     

Ultrastructural comparison of hepatoblastoma and hepatocellular carcinoma.

The ultrastructural characteristics of fetal liver, two hepatoblastomas and two hepatocellular carcinomas were compared. Tumor cells of hepatoblastoma disclosed monotonous nuclei, poorly-developed cytoplasmic membrane system, abundant free ribosomes and prominent glycogen granules. Thos of hepatocellular carcinoma revealed comparatively pleomorphic nuclei, well-developed cytoplasmic membrane system, a few free ribosomes and numerous glycogen granules. Fetal liver showed monotonous nuclei, well-developed RER abundant free ribosomes and prominent glycogen granules. Young mesenchymal cells with well-developed RER and continuous basal lamina surrounding the epithelial cells were detected in both cases of hepatoblastoma but not in those of hepatocellular carcinoma. Tumor cells of hepatoblastoma in a case showed intramitochondrial crystalloids and thick bundles of fibrils in the cytoplasm.     

HSP47在小鼠腭裂和短肢发生过程中的表达

背景与目的:研究热休克蛋白47基因(Heat shock protein 47,HSP47)在小鼠胚胎腭、前肢正常和异常的发育过程中的表达情况。材料与方法:在GD10经口一次分别给予各实验组孕鼠80mg/kg的全反式视黄酸,对照组孕鼠给予等体积的植物油,并分别于GD11~GD18取两组胎鼠的前肢,于GD15~GD17取两组胎鼠的腭,利用RT_PCR方法半定量检测HSP47的表达丰度。结果:HSP47在所有样品中均有表达,其在GD11~GD18正常、异常发育肢的表达均呈随胚龄增大而增加的趋势,对照肢和实验肢分别于GD16、GD17达到高峰,以后基本恒定;其在GD15~GD17正常腭中恒定表达、异常腭中以GD16表达最高;异常肢的表达丰度在GD11~GD18均高于正常肢,正常腭和异常腭的表达丰度在GD15无差异,但在GD16~GD17异常腭的表达丰度则高于正常腭。结论:全反式视黄酸所致的短肢和腭裂中,HSP47的表达呈应激性升高。     

Heat shock protein synthesis and cell survival in clones of normal and simian virus 40-transformed mouse embryo cells

Exposure to hyperthermia induces the synthesis of a set of highly conserved polypeptides known as heat shock proteins (HSPs) in cells of most organisms. Since it has been suggested that these proteins may enhance cell survival by protecting cells from heat-inflicted damage, we studied the synthesis of the major HSPs (Mr 70,000 and 85,000) in clones of normal and SV40-transformed mouse embryo cells. These transformed cells had higher basal HSP levels and consistently synthesized the major HSPs at a higher rate both at physiological temperature and after exposure to heat shock (43-45 degrees). Parallel determination of cell survival showed that the transformed cells were, nevertheless, more susceptible to killing by hyperthermia than were their normal counterparts. Therefore, we conclude that the higher intrinsic resistance of the normal cells to killing by heat is not directly related to basal HSP levels or to the degree to which synthesis of these proteins is induced following exposure to hyperthermia. Considering the abnormal energy metabolism of transformed cells and the known sensitivity of HSP synthesis to energy source restriction, we hypothesize that both basal HSP levels and their induction by heat shock are related to alterations in energy metabolism.     

Hyperthermia and radiation in the treatment of superficial malignancy: an analysis of treatment parameters, response and toxicity

A total of 41 superficial tumours have been treated with radiation alone (21 lesions) or in combination with hyperthermia (20 lesions) in a non-randomized study on 16 patients. A minimum tumour heat dose in excess of 30 min equivalent at 43 degrees C was achieved in 46 of the 80 (57 per cent) hyperthermia treatments. Small lesions received better quality heat treatments and were more likely to achieve a complete response. There is a significant increase in the response of lesions treated with the combined modality compared with radiation alone (P less than 0.01). Similar results were obtained when matched lesions with internal controls were analysed separately. There was an increased incidence of severe skin reactions in the hyperthermia-treated group with the reaction tending to develop more quickly. There were three instances of late fibrosis in the hyperthermia group. There is a significant correlation between the severity of the skin reaction and the average maximum skin heat dose per treatment.     

Hyperthermia exposure impaired the early stage of face recognition: An ERP study

We investigated the effect of hyperthermia exposure on the early stages of face processing by recording event-related potentials (ERPs) elicited by faces and non-face stimuli presented in upright and inverted orientations. Across all conditions, both the peak latencies of P1 and N170 components were earlier in the hyperthermia group than in the control participants. Although no effects of P1 amplitudes were influenced by hyperthermia, the face effect (larger amplitude for faces relative to other object categories) of the N170 was modulated by hyperthermia, whereas the face effect was significant in the control group, it was minimised in the hyperthermia group. The inversion effect of faces on N170 amplitudes, however, was not affected by hyperthermia. These data suggest that the detection of faces in the visual field and their initial streaming to face-specific structural encoding mechanisms are impaired by hyperthermia. However, subsequent face-specific configural processing revealed by the N170 inversion effect is not affected by hyperthermia. In addition, hyperthermia accelerates the early stage of visual perception, regardless of faces or non-face objects.     

Monoclonal antibody to human T-cell leukemia virus p19 defines polypeptide antigen in human choriocarcinoma cells and syncytiotrophoblasts of first-trimester placentas

In previous studies we detected retrovirus RD114 p30-related antigen in human placental syncytiotrophoblasts and antibodies in cord blood sera. We now report that a monoclonal IgG antibody specific for the p19 protein of human T-cell leukemia virus (HTLV) reacts with human syncytiotrophoblastic antigen. When used in the immunoperoxidase tissue-staining procedure, the monoclonal antibody reacted with the syncytiotrophoblastic cells in sections of all early placentas (less than 15 weeks of gestation), blighted ova and benign (hydatidiform mole) or malignant placental tumors (destructive mole, choriocarcinoma). In all cases the reactivity was mainly confined to the cytoplasm of the cells with syncytiotrophoblastic morphology. Normal embryonal or adult tissues tested were negative. In immunoblotting of cultured choriocarcinoma cells, anti-HTLV p19 detected a single polypeptide at mol.wt 28,000 from proteins separated by electrophoresis.     

Reversible downregulation of telomerase activity by hyperthermia in osteosarcoma cells.

Telomerase, a ribonucleoprotein enzyme, maintains telomere length and is expressed by the majority of malignant tumours, but not in normal tissue. Telomerase facilitates the division of tumour cells and its activity has been suggested as a prognostic indicator, but so far the regulation or modulation of telomerase activity has not been described. Hyperthermia has been shown to decrease tumour growth by inhibition of proliferation. Therefore, the effect of hyperthermia on telomerase activity in human osteosarcoma cells was studied. Telomerase activity was measured by the Telomeric Repeat Amplification Protocol (TRAP) assay in three different osteosarcoma cell lines subjected to hyperthermia (42.5 degrees C, 90 min) and in controls cultured under basal conditions (37 degrees C). Telomerase activity was strongly inhibited by hyperthermia and decreased in all cell lines tested after a recovery time of 2 h under basal conditions (37 degrees C) to an activity of approximately 85%, after 12 h approximately 60% and with lowest activity approximately 55% compared to activity of control cells. Telomerase activity then increased and reached the same, i.e. basal, level as before hyperthermia, after 112 h. These results show that hyperthermia results in a reversible downregulation of telomerase activity in osteosarcoma cells. This effect facilities studies on the regulation of telomerase activity and detailed information might lead to new therapeutic strategies.